I have been carrying out some Matrigel invasion assays but in many cases my tumor cells become clumped once they enter in contact with Matrigel. I have tested two Matrigel sources and I even tested different conditions to minimize cell clumping during cell harvest but without success.
Hi Armando
Can you tell me in detail... how you have been performing the experiment?
Clumping of cell usually occurs if TE used to detach cells from plate is not sufficient. Also pipetting matters. Also how have you been seeding the cells on matrigel...i guess after letting matrigel solidify of course...
Please make sure to deactivate trypsin completely before seeding onto matrigel.
Rinse the cells twice with 10% serum containing media and then resuspend in serum free media for plating into matrigel...Hope this helps.
Dear Armando, companies sell pre-coated transwell inserts for that kind of assay (I suppose you use that system) the coating is actually very,very tiny that you can't see it by eye.If you put to much Matrigel, the cells have a much greater thickness to pass and so they can switch to a "migratory" phenotype, being attracted one to the others. That is one of the reason why you get clumps. Another reason could be the cell type you are using, not all the cells have the tendency to be invasive.
Thanks to all for valuable comments. It seems that both incomplete trypsinization and inactication of trypsin were the culprits
Use PBS-EDTA for to detach the cells and add them to 15 ml conical tube containing 4 ml of medium containing 1.0 % BSA.
Collet the cells by centrifugation ( 1200 rpm). Wash the cells one time by resuspending the cell pellet in medium containing 0.1 % BSA.
Hope this helps
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